Humanized anti-prostate stem cell antigen monoclonal antibody

ABSTRACT

Prostate stem cell antigen (PSCA) is expressed in the majority of prostate cancer patients, making it an ideal target for cancer immunotherapy. Murine monoclonal antibody 1G8 binds to PSCA with nanomolar affinity, but its efficacy as a therapeutic agent is limited by the generation of a HAMA response. The present invention discloses humanized 1G8 antibodies in which the majority of the mouse-derived epitopes have been removed. These humanized antibodies bind PSCA with high affinity and specificity, and have been shown to reduce human bladder tumor take in a nude mouse model. These characteristics make the humanized antibodies of the present invention attractive agents for the treatment and detection of tumors expressing PSCA.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional application of U.S. applicationSer. No. 11/432,304, filed May 10, 2006, now U.S. Pat. No. 8,088,908which claims priority to U.S. Provisional Application No. 60/679,848,filed May 10, 2005, the disclosures of which are incorporated byreference herein in their entirety.

GOVERNMENT INTEREST

This invention was made with Government support of Grant number CA043904awarded by the National Institutes of Health. The government has certainrights in this invention.

BACKGROUND

Prostate cancer is the most commonly diagnosed cancer in men, withapproximately 1.7 million men in the U.S. having been diagnosed withthis condition. Over 200,000 new cases of prostate cancer will be addedper year and around 30,000 will die annually, making it the secondleading cause of cancer-related deaths in men. The five-year survivalrate with prostate cancer is 89%, with this percentage jumping to 100%for patients with localized tumors treated with conventional therapeuticapproaches. However, once metastases or hormone-refractory diseasedevelops, therapeutic options are limited. Hence, there is a need todevelop new pharmaceuticals for the treatment of metastatic prostatecancer, as well as a need to identify new diagnostic markers that canbetter discriminate between indolent and aggressive variants of prostatecancer.

Antibody-based therapy using unconjugated, toxin-conjugated orradiolabeled reagents against tumor-associated target antigens hasproven beneficial for solid and hematolymphoid neoplasms (Adams 2005; Wu2005). There are currently 17 monoclonal antibodies (mAbs) approved bythe FDA in the US. Of these, eight (five unconjugated and threeconjugated) are approved for treatment of cancer (Adams 2005). One keyissue with regard to the therapeutic use of monoclonal antibodies hasbeen the response of the human immune system to xenogeneic antibodies.Clinical studies with murine monoclonal antibodies have shown effectivetumor targeting, but have also resulted in rapid clearance of the murineantibody due to the generation of a human anti-murine antibody (HAMA)immune response (Schroff 1985; Shawler 1985). The present inventionprovides humanized antibodies for use in the diagnosis and treatment ofprostate cancer with minimal HAMA response.

SUMMARY

In certain embodiments, a humanized antibody is provided that combines ahuman or humanized immunoglobulin framework with a binding site thatrecognizes the same epitope as murine monoclonal antibody 1G8. Incertain embodiments, the humanized antibody may bind PSCA with a K_(A)of at least about 2.5×10⁸. In certain embodiments, the frameworkimmunoglobulin may be a humanized antibody 4D5 version 8 in which one ormore of the following residues have been replaced with equivalentresidues from murine 1G8 monoclonal antibody (residues numberedaccording to Kabat system): L4, L24-L34, L46, L50-L56, L66, L70, L71,L89-L97, H26-H35, H48, H49, H50-H59, H60-H65, H66, H67, H69, and/orH93-H102. In these embodiments, the light chain variable region of thehumanized antibody may have the amino acid sequence set forth in SEQ.ID. NO:7 or SEQ. ID. NO: 37 and the heavy chain variable region setforth in SEQ. ID. NO:8, SEQ. ID. NO:9, or SEQ. ID. NO:38. In certainembodiments, the humanized antibody may be associated with a conjugatesuch as a toxin, a cytokine, a chemotherapeutic agent, or a radiolabel.

In certain embodiments, an isolated polynucleotide is provided encodinga humanized antibody that combines a human or humanized immunoglobulinframework with a binding site that recognizes the same epitope as murinemonoclonal antibody 1G8. In certain embodiments, the variable lightchain of the humanized antibody may have the amino acid sequence setforth in SEQ. ID. NO:7 or SEQ. ID. NO:37, and the variable heavy chainof the humanized antibody may have the amino acid sequence set forth inSEQ. ID. NO:8, SEQ. ID. NO:9, or SEQ. ID. NO:38. In certain embodiments,the polynucleotide may include the sequence set forth in SEQ. ID. NO:29,while in other embodiments it may include the sequence set forth in SEQ.ID. NO:30. In certain embodiments, the polynucleotide may be part of avector, and in certain of these embodiments the vector may be containedwithin a host cell.

In certain embodiments, a method is provided for treating a tumor thatexpresses PSCA by administering to a subject a humanized antibody thatcombines a human or humanized immunoglobulin framework with a bindingsite that recognizes the same epitope as murine monoclonal antibody 1G8.In certain embodiments, the humanized antibody may be bound to aconjugate such as a toxin, a cytokine, a chemotherapeutic agent, or aradiolabel. In certain embodiments, the tumor being treated may be abladder tumor or a prostate tumor.

In certain embodiments, methods are provided for detecting or localizinga tumor that expresses PSCA by administering to a subject a radiolabeledhumanized antibody that combines a human or humanized immunoglobulinframework with a binding site that recognizes the same epitope as murinemonoclonal antibody 1G8, and scanning the subject with a photoscanner todetect the activity of the humanized antibody.

In certain embodiments, a composition is provided for detecting,localizing, or treating a tumor that expresses PSCA that includes ahumanized antibody combining a human or humanized immunoglobulinframework with a binding site that recognizes the same epitope as murinemonoclonal antibody 1G8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Sequence alignment of 1G8 variable light (V_(L)) and variableheavy (V_(H)) domains with the 1QOK modeling template.

FIG. 2: Superimposed alpha carbon traces of 1G8 (black) and 4D5 version8 (gray) framework regions. Two orthogonal views (A and B) are shown.

FIG. 3: Biochemical and functional characterization of purified 1G8mAbs. A. SDS-polyacrylamide gel electrophoresis of purified mu1G8(lane 1) and ch1G8 (lane 2) under non-reducing and reducing conditions.B. Purified mu1G8 and ch1G8 were assayed by flow cytometry for bindingto LNCap-PSCA cells and detected with Alexa-488 conjugated anti-mouseand anti-human IgG antibodies. C. Western blot to demonstrate specificbinding of ch1G8 to PSCA. Lane 1=GST-PSCA; Lane 2=293T negative controllysate and; Lane 3=293T-PSCA. The blot was incubated with ch1G8 andanti-human IgG AP-conjugated antibodies were used for detection.Molecular weight standards appear next to lane 3. D. SDS-polyacrylamidegel electrophoresis of purified mu1G8 (lane 1), hu1G8-A (lane 2) andhu1G8-B (lane 3) under non-reducing conditions and reducing conditions.E. Flow cytometry. Purified mu1G8, hu1G8-A and hu1G8-B were assayed forbinding to LNCap-PSCA cells and detected with Alexa-488 conjugatedanti-mouse and anti-human IgG antibodies. F. Competitive ELISA bindingassay using biotinylated intact mu1G8 monoclonal antibody as tracer andincreasing amounts of unlabeled competitors, i.e., mu1G8, hu1G8-A andhu1G8-B.

FIG. 4: Ribbon rendering of hu1G8-A (A) and hu1G8-B (B) models. Blacksegments represent human residues taken from 4D5 version 8 coordinates.White segments represent murine residues taken from 1G8 coordinates.Segment boundaries are labeled with residue numbers. Residues renderedin ball-and-stick are framework residues that were back mutated to theirmurine counterparts.

FIG. 5: Structure-based sequence alignment of mu1G8 variable light(V_(L)) and variable heavy (V_(H)) domains with 4D5 version 8(Herceptin), hu1G8-A, and hu1G8-B. Peptide segments of murine origin areshown in bold typeface. Sequences and CDR boundaries are numberedaccording to Kabat. Note that the segments that were transplanted fromdonor to acceptor to create hu1G8-A and hu1G8-B model do not necessarilycorrespond to Kabat CDRs.

FIG. 6: Coronal microPET scan images (4 mm thick slices) and microPET/CTfused images of ¹²⁴I-labeled anti-PSCA antibodies in individual SCIDmice bearing LAPC-9 (antigen positive) and PC3 (antigen negative)xenografts at 72 hours and 168 hours. A. Coronal view of a SCID mousegiven 127 μCi 124I-mu1G8. B. Coronal view of a SCID mouse given 110 μCi¹²⁴I-hu1G8-B. The negative tumor is indicated by an arrow in themicroPET/CT fused images.

FIG. 7: Inhibition of tumor take of SW780 bladder carcinoma cells inSCID mice by mu1G8 and hu1G8-B (clone 2B3).

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

DEFINITIONS

The term “antibody” as used herein refers to any immunoglobulin,including antibodies and fragments thereof, that binds to a specificantigen. The term encompasses polyclonal, monoclonal, chimeric,humanized, and bispecific antibodies, and may refer to an intactimmunoglobulin molecule or to some immunologically active portion of animmunoglobulin molecule, such as a Fab, Fab′, F(ab′)₂, or Fv portion. Acomplete antibody comprises two heavy chains and two light chains. Eachheavy chain consists of a variable region and a first, second, and thirdconstant region, while each variable chain consists of a variable regionand a constant region. The antibody has a “Y” shape, with the stem ofthe Y consisting of the second and third constant regions of two heavychains bound together via disulfide bonding. Each arm of the Y consistsof the variable region and first constant region of a single heavy chainbound to the variable and constant regions of a single light chain.

The phrase “humanized antibody” as used herein refers to agenetically-engineered antibody wherein the variable region comprisesthe CDRs or portions of the CDRs of a non-human antibody and theframework regions of a human antibody, and the constant region comprisesthe constant region of a human antibody.

The term “epitope” as used herein refers to the specific group of atomson an antigen molecule to which a specific antibody binds, causing animmune response.

The term “acceptor” as used herein refers to a molecule that providesthe structural framework for creation of a humanized molecule, such as ahuman immunoglobulin.

The term “donor” as used herein refers to the molecule that provides thebinding site element of a humanized molecule. This molecule is generallya non-human polypeptide, such as a murine monoclonal antibody.

The term “polypeptide” as used herein refers to any peptide or proteincomprising two or more amino acids joined to each other by peptide bondsor modified peptide bonds, i.e., peptide isosteres. “Polypeptide” refersto both short chains, commonly referred to as peptides, oligopeptides,or oligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the gene-encoded aminoacids. “Polypeptides” include amino acid sequences modified by naturalprocesses, such as posttranslational processing, or by chemicalmodification using techniques that are well known in the art. Suchtechniques have been described in basic texts, more detailed monographs,and voluminous research literature. Modifications can occur anywhere ina polypeptide, including the peptide backbone, the amino acidside-chains, and the amino or carboxyl termini. One skilled in the artwill recognize that the same type of modification may be present in thesame or varying degrees at several sites in a single polypeptide, andthat a single polypeptide may contain multiple modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched, and branchingcyclic polypeptides may result from posttranslational natural processes,or they may be created by synthetic methods. Polypeptide modificationsinclude acetylation, acylation, ADP-ribosylation, amidation, attachmentof an antibody Fc domain (native, recombinant, or humanized),biotinylation, carboxymethylation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of a lipid orlipid derivative, covalent attachment of a nucleotide or a nucleotidederivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, dansylation, demethylation, disulfide bond formation,enzyme labeling, farnesylation of cysteine residues, FITC-conjugation,formation of covalent cross links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, phosphorylation, prenylation,proteolytic processing, racemization, radiolabeling, selenoylation,succinylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. See, for example,Proteins—Structure and Molecular Properties, 2^(nd) Ed., T. E.Creighton, W.H. Freeman and Company, New York, 1993; Wold, F.,“Posttranslational Protein Modifications: Perspectives and Prospects,”pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C.Johnson, Ed., Academic Press, New York, 1983; Seifter, S., Englard, S.1990. Analysis for protein modifications and nonprotein cofactors. MethEnzymol 182:626-646; Rattan, S. I., Derventzi, A., Clark, B. F. 1992.Protein synthesis: posttranslational modifications and aging. Ann N YAcad Sci 663:48-62.

The term “polynucleotide” as used herein refers to anypolyribonucleotide, polydeoxyribonucleotide, or hybridpolyribo-polydeoxyribonucleotide, including naturally occurringpolynucleotides, synthetic polynucleotides, or any chemically,enzymatically, or metabolically modified forms of naturally occurringpolynucleotides. Polynucleotides may contain any of the standardpyrimidine or purine bases (i.e., adenine, guanine, cytosine, thymine,uracil), as well as any modified or uncommon bases such as tritylatedbases or inosine. In addition, the backbone of a polynucleotide may bemodified for stability or for other reasons.

The phrase “coding sequence” as used herein refers to a polynucleotidesequence having sequence information necessary to produce a gene productfor which expression is desired, according to normal base pairing andcodon usage relationships. In order to produce this gene product, thecoding sequence must be placed in such relationship to transcriptionalcontrol sequences (possibly including control elements and translationalinitiation and termination codons) that a proper length transcript willbe produced and will result in translation in the appropriate readingframe to produce a functional desired product.

The term “isolated” as used herein means altered “by the hand of man”from the natural state. If an “isolated” composition or substance occursin nature, it has been changed or removed from its original environment,or both. For example, a polynucleotide or a polypeptide naturallypresent in a living animal is not “isolated,” but the samepolynucleotide or polypeptide is “isolated” if it has been sufficientlyseparated from the coexisting materials of its natural state so as toexist in a substantially pure state. “Isolated” as used herein does notexclude artificial or synthetic mixtures with other compounds ormaterials, or the presence of impurities that do not interfere withactivity.

The term “vector” as used herein refers to a vehicle into which apolynucleotide encoding a protein may be operably inserted so as tobring about the expression of that protein. A vector may be used totransform, transduce, or transfect a host cell so as to bring aboutexpression of the genetic element it carries within the host cell.Examples of vectors include plasmids, phagemids, cosmids, and artificialchromosomes such as yeast artificial chromosome (YAC), bacterialartificial chromosome (BAC), or P1-derived artificial chromosome (PAC),bacteriophages such as lambda phage or M13 phage, and animal viruses.Categories of animal viruses used as vectors include retrovirus(including lentivirus), adenovirus, adeno-associated virus, herpesvirus(e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, andpapovavirus (e.g., SV40). A vector may contain a variety of elements forcontrolling expression, including promoter sequences, transcriptioninitiation sequences, enhancer sequences, selectable elements, andreporter genes. In addition, the vector may contain an origin ofreplication. A vector may also include materials to aid in its entryinto the cell, including but not limited to a viral particle, aliposome, or a protein coating.

The phrase “host cell” as used herein refers to a cell into which avector has been introduced. A host cell may be selected from a varietyof cell types, including for example bacterial cells such as E. coli orB. subtilis cells, fungal cells such as yeast cells or Aspergilluscells, insect cells such as Drosophila S2 or Spodoptera Sf9 cells, oranimal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLacells, BHK cells, HEK 293 cells, or human cells.

The phrase “scanning device” as used herein refers to any device fordetecting a radionuclide or fluorescent agent, such as a photoscannerfor detecting radioactive activity. More specifically, “scanning device”refers to a device capable of detecting the presence of a radionuclidethat has been injected in a subject, identifying the specific locationof the radionuclide within the subject, and quantifying the amount ofradionuclide within that specific location.

Abbreviations

The following abbreviations are used herein: CDR, complementaritydetermining region; HAMA, human anti-murine antibody; mAb, monoclonalantibody; PSCA, prostate stem cell antigen; SCID, severe combined immunedeficient; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gelelectrophoresis; V_(H), variable heavy; V_(L), variable light.

Humanized 1G8 Antibodies

Prostate cancer is an attractive target for antibody-based therapy forseveral reasons: 1) tissue-specific rather than tumor-specific targetingis allowed as the prostate is a non-essential organ; 2) metastases areusually small enough to enable good penetration; 3) the metastases thatprimarily involve bone marrow and lymph nodes are in locations thatreceive high levels of circulating antibody; 4) therapeutic effect canbe rapidly monitored by serum prostate-specific antigen (PSA); and 5)prostate cancer is radiation sensitive, rendering it an excellent targetfor radiolabeled antibody therapies.

Several mAbs targeting general tumor antigens or prostate specificantigens have been developed that are in preclinical and clinicaldevelopment (Maynard 2000; Smith-Jones 2004). However, human anti-mouseantibody (HAMA) responses, dose limiting toxicity, and low therapeuticefficacy are issues that have been reported.

One solution to HAMA response problem is to generate human antibodiesfrom human immunoglobulin phage display libraries (Winter 1994) ortransgenic animals (Bruggemann 1991, Mendez 1997). These techniques haveproduced a small yet growing number of antibodies with high specificityand affinity. However, antibodies produced by these methods have eitherexhibited specificity only for immobilized antigen or have exhibitedpoor expression as intact antibodies in mammalian cell culture. Anothersolution to the HAMA response problem has been the use of recombinantmethodologies to generate chimeric monoclonal antibodies, whichgenerally consist of a murine antigen-binding variable domain coupled toa human constant domain. These chimeras have a lower frequency of immuneresponse, but they are not effective for all antibodies and may stillgenerate an immune response against the murine variable region. A thirdsolution to the HAMA response problem is the utilization of humanized orreshaped monoclonal antibodies. These consist of human antibodies inwhich only the complementarity determining region (CDR) has beensubstituted with an animal CDR.

The current generation of humanized monoclonal antibodies that have beenapproved for therapy are the result of grafting murine-derived CDR'sonto a human antibody framework (Jones 1986; Low 1986). This process ofCDR-grafting is a well established technique, but it has a downside inthat it frequently generates an antibody with substantially decreasedantigen binding affinity compared to the parental antibody. Thisdecreased affinity is the result of unanticipated steric clashes betweenthe human immunoglobulin framework and the murine CDR side chains, whichalter the CDR loop conformation. This disadvantage can be overcome bythe reiterative process of back-mutagenesis, which involves therestoration of key murine framework residues that are responsible formaintaining the correct CDR loop conformations (Foote 1992). However,this process is laborious and random.

There are many examples in the published literature of antibodyhumanization via CDR-grafting (O'Brien 2003). However, this processoften results in an antibody with substantially decreased bindingaffinity compared to the parental antibody. This decreased affinity iscaused by unanticipated steric clashes between the human immunoglobulinframework and the mouse CDR residues, which alter the conformation ofthe antigen binding loops. Such steric clashes can be overcome byintroducing back-mutations to restore key murine framework residuesresponsible for correct loop conformation, but this process is laboriousand often reiterative (Foote 1992). The reason steric clashes inhumanized antibodies have been unanticipated is because the constructshave been designed using molecular models of the graft donor and graftacceptor molecules, rather than actual crystal structures of one or bothof those molecules. For instance, donor and acceptor molecules havepreviously been selected based on their amino acid sequences (U.S. Pat.Nos. 5,639,641; 6,639,055). In these approaches, the amino acidsequences of exposed regions of the murine antibody are obtained, andsequence databases are used to select a human antibody with the same ora similar sequence.

Prostate stem cell antigen (PSCA) is a predominantly prostate-specificcell surface antigen that is expressed in the majority of prostatecancer patients (Reiter 1998; Gu 2000; Reiter 2000). PSCA is acysteine-rich 123 amino acid glycosylphosphatidylinositol (GPI)-anchoredsurface glycoprotein related to the Thy-1/Ly-6 family (Reiter 1998).Studies have shown PSCA expression in 80% of local disease and in allbone metastatic lesions examined (Reiter 1998; Gu 2000). Elevated PSCAexpression has been correlated with increased tumor stage, grade, andprogression to androgen dependence (Gu 2000). The high expression levelof PSCA in cancerous tissue, combined with its low expression in normaltissue, makes it an ideal target for immunotherapy.

Several antibodies were raised previously against a PSCA-GST fusionprotein (Gu 2000). Five of these antibodies (1G8, 4A10, 3E6, 3C5, and2H9) were subcloned, purified, and shown to bind PSCA by flow cytometry.Their epitope localization on PSCA was determined and classified intothree regions: 4A10, 2H9, and 3C5 bound to a region close to theN-terminus (aa 21-50); 1G8 bound to the middle region (aa 46-85), and3E6 bound to a region close to the C-terminus (aa 85-99). Two of theseanti-PSCA mAbs, 1G8 (IgG1κ; K_(D)=1 nM) and 3C5 (IgG2aκ; K_(D)=43 nM)were examined for their inherent anti-tumor efficacy in subcutaneous andorthotopic prostate cancer xenografts models (Saffran 2001). Althoughboth antibodies demonstrated similar anti-tumor activity, 1G8 wasconsistently more efficacious than 3C5 in growth retardation oforthotopic tumors, which lead to greater prolongation of survival. Thissuperiority was attributed to higher affinity, isotype, and/or thelocalization of the epitope on PSCA. However, it has recently been shownthat F(ab′)₂ alone can exert this effect, suggesting that Fc is notrequired (Gu 2005).

Biodistribution studies of 1G8 radiolabeleled with ¹¹¹In are presentedherein, and specific tumor targeting is demonstrated. However, theutility of 1G8 in human immunotherapy is likely to be limited due toimmunogenicity and the potential of the antibody to generate a HAMAresponse. Thus, chimeric and humanized 1G8 antibodies have beendeveloped in order to generate 1G8 with substantially reducedimmunogenicity.

Murine 1G8 variable genes were isolated and fused to human κ and IgG1constant genes to produce anti-PSCA mouse-human chimeric 1G8 (ch1G8).Although ch1G8 retained the binding specificity of parental antibody anddemonstrated improved in vivo efficacy over mu1G8 in a prostate cancermodel, it expressed at low levels and was unstable in PBS. Since it iswell known that chimeric antibodies can induce an immune response inhumans when administered repeatedly, a humanized mu1G8 antibody wasgenerated to improve stability and expression and reduce immunogenicity.

Humanized 1G8 (hu1G8) was generated using a CDR grafting strategy. Sincethe crystal structure of 1G8 was not yet available, a molecular model ofthe Fv region of 1G8 was created using the anti-CEA antibody MFE-23 (PDBfile 1QOK) as a template. 1QOK was chosen as the sole modeling templatebecause it shares high sequence identity with 1G8, five of its sixhypervariable loops are of the same length as those of 1G8, and the twochains form a cognate pair, which greatly increases the accuracy of themodeled V_(L):V_(H) interface.

Antibody 4D5 version 8 (hu4D5v8) was chosen as a suitable frameworkprovider based largely on its remarkably low immunogenicity: only 0.5%of breast cancer patients participating in a large-scale multinationaltrial developed HAHA (Cobleigh 1999). This phenomenon may be related tothe fact that the V_(H) and V_(L) regions of 4D5v8 are derived from thetwo most common human germline gene families, namely the V_(H)3 family,which constitutes 43% of all human V_(H) germline genes, and the Vκ1family, which constitutes 25% of all human V_(L) germline genes (Ewert2003). In addition, the 4D5v8 framework has been extensivelycharacterized, is known to be stable, and exhibits a high degree ofstructural similarity to the molecular model of 1G8 discussed above.

Previous studies have used 4D5v8 as a framework provider forhumanization of the anti-CEA mAb T84.66 (Yazaki 2004). In those studies,two versions (A and B) of the construct were created because it wasuncertain whether the C-terminal end of CDR-H2 (H60-H65) made contactwith antigen and therefore had to be carried over into the graft. ForhuT84.66, the identity of residues in this region proved to beunimportant; when purified and assayed, both versions exhibited the sameaffinity (Yazaki 2004). For mu1G8, it was reasonable to assume theopposite, i.e., that these residues might be critical for antigenrecognition since CDR-L1 and CDR-H3 are both truncated, leaving CDR-H2highly exposed.

Two humanized 1G8 antibodies, hu1G8-B (M6B) and hu1G8-A (M6A), werecreated. In both hu1G8-B and hu1G8-A, six peptide segments correspondingapproximately with CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3were deleted from the hu4D5v8 framework molecule and replaced withcorresponding murine peptide segments from 1G8. With regards to thesesegments, the only difference between hu1G8-A and hu1G8-B was five extraCDR-H2 residues replaced with their murine equivalents in hu1G8-B. Inhu1G8-B, two additional hu4D5v8 residues were replaced with their murineequivalent to minimize steric hindrance between donor and acceptor sidechains, while in hu1G8-A only one of these residues was replaced withits murine equivalent. In both hu1G8-B and hu1G8-A, four additionalhu4D5v8 residues were replaced with their murine equivalent based onpotential effects on antigen binding.

Fully synthetic genes encoding the V_(L) chain and V_(H) chains ofhu1G8-A and hu1G8-B were created using splice overlap extensionpolymerase chain reaction. Purified full-length V_(L) chain genes wereligated into the expression plasmid pEE12, while V_(H) chain genes wereligated into the expression plasmid pEE6. Both of these plasmids hadbeen previously modified to contain the cDNA sequence of thecorresponding constant regions of a human IgG₁ antibody. The heavy chaingene was then removed from pEE6 and ligated into the pEE12 light chainplasmid. This dual chain pEE12/6 plasmid was electroporated into murinemyeloma NS0 cells, and transfectants were screened for secretion ofhumanized 1G8 using ELISA. The hu1G8-B and hu1G8-A clones with thehighest expression levels were expanded. SDS-PAGE analysis of purifiedaliquots of hu1G8-B and hu1G8-A under reducing conditions revealed twobands corresponding to the light and heavy chain polypeptides, whileELISA and FACS analysis confirmed that both humanized antibodiesspecifically bound PSCA. The hu1G8-B antibody, which includes moremurine residues in CDR-H2 than hu1G8-A, demonstrated a six-fold higherrelative binding affinity to PSCA compared to hu1G8-A. Thus, it appearsthat for this particular antibody, the C-terminal end of CDR-H2 makescontact with antigen.

Although the binding affinity of hu1G8-B for PSCA was about five-foldlower than that of mu1G8, its ability to target tumor was equivalentwhen evaluated by microPET imaging. In addition, studies in nude micewith bladder carcinoma suggest that the in vivo efficacy of hu1G8 issuperior to that of the parental mu1G8. It has recently been shown thatmu1G8 acts by a direct Fc-independent mechanism to inhibit prostatetumor growth both in vivo and in vitro (Gu 2005). However, since nudemice have some immune effector cells, engagement of the mouse immunesystem such as antibody dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC) brought on by switching frommouse to human IgG1 isotype may also play a factor in inhibiting tumorgrowth in this model. The lower activity of both antibodies in the tumorat a later time point (168 hours vs. 94 hours i.e. at the time ofsacrifice in the two microPET studies) is probably due to the activeinternalization of the 1G8. Upon internalization, radioiodinatedantibodies become proteolytically degraded into iodotyrosines that arerapidly deiodinated resulting in fast disappearance of the radioactivity(Geissler 1992; Xu 1997).

The 1G8 molecular model was further refined using the crystal structuresof additional antibodies with CDR loops very similar or identical tothose of 1G8. Based on the overlap of this refined model with hu4D5v8,six additional hu1G8-B residues were selected for back mutation to thecorresponding murine residue to create hu1G8-C (M6C). The purpose ofthese back mutations was to increase the PSCA binding affinity of thehumanized antibody to that of the murine 1G8 antibody.

The humanized antibodies of this invention may be conjugated with smallmolecule toxins, cytokines, or chemotherapeutic agents (e.g.,doxurubicin) for specific delivery to cancer cells. In addition, bindingof the humanized antibodies to tumor cells may be used to recruit hostimmune responses. This host immune response may be increased byutilizing bivalent antibodies, with one binding site corresponding tothe humanized construct of the present invention and another bindingsite that recognizes cytotoxic T-cells.

The humanized antibody of the present invention may be administered fordetection or treatment of PSCA expressing tumors by subcutaneous,peritoneal, intravascular, intramuscular, intradermal or transdermalinjection, among other methods. The antibody may be labeled with avariety of labeling agents, including radioactive labels such as iodine(¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I), sulfur (³⁵S), tritium (³H), indium (¹¹¹In,¹¹²In), carbon (¹⁴C), copper (⁶⁴Cu, ⁶⁷Cu), yttrium (⁸⁶Y, ⁸⁸Y, ⁹⁰Y),lutitium (¹⁷⁷Lu), other lanthanides, luminescent labels, or fluorescentlabels, or some combination thereof. Those of skill in the art willrecognize that a variety of conjugates may be coupled to the humanizedantibody (see, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds.),Carger Press, New York, (1989)). These conjugates may be linked to thehumanized antibody by covalent binding, affinity binding, intercalation,coordinate binding, or complexation, among other methods. Conjugates mayalso consist of chemotherapeutic agents such as vindesine, cisplatin,doxurubicin, or adriamycin, or any other compound useful in thetreatment of cancer, or toxins such as ricin or diptheria toxin, amongothers.

For detection and localization of tumors expressing PSCA, the humanizedantibody of the present invention will be administered at a dosesufficient for detection by a scanning device. This dosage will bedependent on the type of label being used. The type of scanning deviceto be used will vary depending on the label being used, and one skilledin the art will easily be able to determine the appropriate device.

For treatment of tumors expressing PSCA, the humanized antibody of thepresent invention may be prepared at an effective dose as a formulationwithin pharmaceutically acceptable media. This formulation may includephysiologically tolerable liquids, gels, solid carriers, diluents,adjuvants, or excipients, or some combination thereof. Thepharmaceutical formulation containing the humanized antibody may beadministered alone or in combination with other known tumor therapies.Effective dosage will depend in part on the weight, age, and state ofhealth of the subject, as well as the administration route and extent oftumor development.

The humanized antibody of the present invention or portions thereof maybe expressed using any appropriate expression system. Polynucleotidesencoding variable light (V_(L)) and variable heavy (V_(H)) chains of thehumanized antibody may be expressed using separate vectors, or bothchains may be expressed from one vector. Suitable vectors may contain avariety of regulatory sequences, such as promoters, enhancers, ortranscription initiation sequences, as well as genes encoding markersfor phenotypic selection. Such additional sequences are well known inthe art. Additionally, the vector may contain a polynucleotide sequenceencoding the constant regions of the heavy chain (C_(H)) and light chain(C_(L)) of a human immunoglobulin. Alternatively, the vector may expressonly the V_(H) and V_(L) chains of the humanized antibody, with theexpressed polypeptide comprising an Fv fragment rather than a wholeantibody.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention.

EXAMPLES Example 1 Construction and Expression of Chimeric 1G8 (ch1G8)

ch1G8 was constructed using a procedure similar to that describedpreviously by Coloma et al. (Coloma 1992). mu1G8 hybridoma cells (Gu2000) from American Tissue Culture Collection (ATCC No. HB-12612,deposited Dec. 11, 1998; ATCC, Manassas, Va.) were grown in Iscove'sModified Dulbecco's Medium (IMDM)/5% FBS. Total RNA was extracted fromthe cells using ULTRASPEC® Total RNA Isolation Reagent (BiotecxLaboratories, Houston, Tex.). First strand DNA synthesis (cDNA) wasprepared from total RNA using GENEAMP® RNA PCR kit and oligo d(T)₁₆ asprimer (Roche Applied Biosystems, Foster City, Calif.). The variableheavy (V_(H)) and light (V_(L)) chain genes were amplified from cDNAusing primer sequences described by Coloma et al. (Coloma 1992). PCRproducts were cloned into pCR2.1 (Invitrogen Life Technologies,Carlsbad, CA) and several clones were sequenced. In order to verify thatthe sequences were correct, the primary amino acid sequences werecompared to tryptic peptide sequences of the intact, parental mu1G8 mAbheavy and light chains as described (Wu 2001).

The mu1G8 V_(L) gene was cloned into the human κ light chain expressionvector pAG4622 using EcoRV and SalI restriction sites. The mu1G8 V_(H)gene was cloned into the human IgG1 heavy chain expression vectorpAG3021 via EcoRV and NheI restriction sites. Linearized 1G8 light chainvector was electroporated into P3×63.Ag8.653 non-producing myeloma cellsgrown in Iscove's Modified Dulbecco's Medium (IMDM)/5% FBS. Cells wereselected in media supplemented with xanthine, mycophenolic acid andhypoxanthine (XMH). High expressing clones were identified by ELISA ofthe culture supernatants using goat anti-human κ alkalinephosphate-conjugated antibodies (Jackson ImmunoResearch Laboratories,West Grove, Pa.). Cells expressing high levels of ch1G8 light chain wereelectroporated with linearized ch1G8 heavy chain vector and selected inpresence of histidinol. Secretion of ch1G8 to the cell culturesupernatant was measured by ELISA using goat anti-human IgG (Fcspecific) to capture ch1G8 constructs andalkaline-phosphatase-conjugated goat anti-human IgG (Fc specific)antibodies (both from Jackson ImmunoResearch Laboratories) fordetection. The best producing clones were further evaluated by Westernblot for size verification. High producing clones were expanded intoTripleFlasks (Nalge Nunc Int'l, Rochester, N.Y.) or into CELL-PHARM® 100hollow fiber cell culture system (Unisyn Technologies, Hopkinton, Mass.)in 1MDM/1% FBS supplemented with GLUTAMAX® (Invitrogen LifeTechnologies). The expression level of ch1G8 in the highest producingclone was only about 2-3 μg/mL in TripleFlasks.

Example 2 Development of a Murine 1G8 Humanization Design

Homology modeling preceded humanization since no crystal structure wasavailable for the mu1G8 Fv region. A crystallographic template suitablefor modeling this region was selected by performing a sequence-basedsearch of the Protein Data Bank (Berman 2002) using the FASTA program(Pearson 1988). Anti-CEA antibody MFE-23 (PDB file 1QOK) (Boehm 2000)was chosen as a template for modeling 1G8 for three reasons: 1) thepercent sequence identity was high (81% for V_(L), 80% for V_(H)); 2)with the exception of the CDR-H3 loop, the lengths of the hypervariableloops were identical and oftentimes the actual sequences were identicalor nearly identical; and 3) the V_(L) and V_(H) templates formed acognate pair. The amino acid sequences of the V_(L) and V_(H) chains of1G8 are set forth in SEQ. ID. NOs: 1 and 2, respectively, while theamino acid sequence of the V_(L) and V_(H) chains of 1QOK are set forthin SEQ. ID. NOs: 3 and 4, respectively. The amino acid sequencealignment of the V_(L) and V_(H) chains of 1G8 and 1QOK is shown in FIG.1.

The HOMOLOGY module within INSIGHT II® (Accelrys, San Diego, Calif.) wasused to construct the molecular model. CDR-H3 was modeled by deletingresidues H96-H100c from the template and forming a peptide bond betweenresidues H95 and H101.

After repositioning the side chains of residues L46, L90, and L95 toeliminate steric clashes, the stereochemistry of the model was improvedusing conjugate gradients energy minimization until the maximumderivative was less than 5 kcal/mol-Å.

CDR-grafting was selected as a suitable strategy for humanizing 1G8(Jones 1986; Low 1996). Humanized antibody 4D5 version 8 (hu4D5v8,anti-p185^(HER2), HERCEPTIN® (Trastuzumab), PDB file 1FVC) (Eigenbrot1993) was selected as the most appropriate framework provider for theproposed CDR graft based on previous success using this Fv forhumanization (Yazaki 2004). The degree of overlap between hu4D5v8 andthe 1G8 model was very high (root-mean-square deviation of 1.07 Å for1320 backbone atoms), and the angle of V_(L)-V_(H) domain pairing wasessentially the same as seen from two orthogonal views of thesuperimposed alpha carbon traces in FIG. 2. The amino acid sequences ofthe V_(L) and V_(H) chains of hu4D5v8 are set forth in SEQ. ID. NOs: 5and 6, respectively.

Using the HOMOLOGY module, the Kabat CDRs of the 1G8 model were graftedonto the hu4D5v8 framework. The CDR graft strategy is shown by sequencealignment in FIG. 5. Visual inspection of the superimposed structuressuggested that minimal disruption of the CDR loops could be achieved bydeleting six peptide segments from the hu4D5v8 graft acceptor structure(Kabat residues L24-L34, L50-L56, L89-L97, which correspond to residues24-34, 50-56, and 89-97 of SEQ. ID. NO:5, and Kabat residues H26-H35,H50-H65, and H93-H102, which correspond to residues 26-35, 50-66, and97-109 from SEQ. ID. NO:6) and replacing them with six correspondingpeptide segments from the mu1G8 graft donor structure (Kabat residuesL24-L34, L50-L56, L89-L97, which correspond to residues 24-33, 49-55,and 88-96 of SEQ. ID. NO:1, and Kabat residues H26-H35, H50-H65, andH93-H102, which correspond to residues 26-35, 50-66, and 97-101 of SEQ.ID. NO:2).

Upon completing the graft, the resulting molecular model was inspectedfor potential steric clashes between donor and acceptor side chains atthe CDR-framework interface. A clash between hu4D5v8 framework residueL4 (methionine, residue 4 in SEQ. ID. NO:5) and mu1G8 CDR-L1 residue L33(isoleucine, residue 32 in SEQ. ID. NO:1) was alleviated by replacingthe hu4D5v8 L4 residue with the L4 residue from 1G8 (leucine, residue 4in SEQ. ID. NO:1). A clash between hu4D5v8 framework residue H67(phenylalanine, residue 68 in SEQ. ID. NO:6) and 1G8 CDR-H2 residue H63(phenylalanine, residue 64 in SEQ. ID. NO:1) was alleviated by replacingthe hu4D5v8 H67 residue with the H67 residue from 1G8 (alanine, residue68 in SEQ. ID. NO:1).

The molecular model of hu1G8 revealed an unusual binding site topology,namely a cleft arising from a truncated CDR-L1 loop having ten residuesrather than 11 and a severely truncated CDR-H3 loop having just threeresidues (FIG. 5). Because of these loop truncations and the fact thatthe tip of the CDR-H3 loop lacked side chains (sequence Gly-Gly), fourframework residues gained the potential to interact with antigen: L46(Arg), L66 (Gly), H93 (Lys), and H94 (Thr). All four of these residueswere replaced with their murine equivalent in the humanized model. Theresulting humanized structure, hu1G8-B, was subjected to an energyminimization algorithm (conjugate gradients to a maximum derivative of5.0 kcal/mol-Å) to optimize bond lengths and angles at the splicejunctions.

The binding site in the final model suggested that the C-terminal end ofKabat CDR-H2 (residues H60 to H65), which does not frequently makecontact with antigen (Padlan 1995; MacCallum 1996), might indeedparticipate in antigen binding since CDR-L1 and CDR-H3 are truncated.For this reason, a second humanized 1G8, hu1G8-A, was created. Inhu1G8-A, residues H60-H65 were treated as framework residues, meaningthat they retained the amino acid sequence of hu4D5v8 (residues 61-66 ofSEQ. ID. NO:6) rather than being replaced with the correspondingresidues from 1G8. hu1G8-A also retains the hu4D5v8 framework residue atH67 (phenylalanine) that was replaced with alanine in hu1G8-B.

The amino acid sequence of the V_(L) chain of hu1G8-A and hu1G8-B is setforth in SEQ. ID. NO:7. The amino acid sequences of the V_(H) chains ofhu1G8-A and hu1G8-B are set forth in SEQ. ID. NOs. 8 and 9,respectively. The structure-based sequence alignment of 1G8 V_(L) andV_(H) chains with hu4D5v8 (HERCEPTIN®), hu1G8-A, and hu1G8-B is shown inFIG. 5. A ribbon rendering of hu1G8-A and hu1G8-B is set forth in FIG.4.

Example 3 Construction of Synthetic Genes Encoding hu1G8

Splice overlap extension polymerase chain reaction (SOE-PCR) (Horton1989) using multiple overlapping oligonucleotides was used to synthesizehu1G8 V_(L) and V_(H) genes as previously described (Yazaki 2004). Eightoligonucleotides (Integrated DNA Technologies, Inc., Coralville, Iowa),ranging in size from 62 to 89 nucleotides, were required for each domainconstruct. The degree of overlap between adjacent oligonucleotidescorresponded to 30 base pairs. The PCR primer sequences for the variablelight (V_(L)) and variable heavy (V_(H)) domains are set forth in SEQ.ID. NOs:10-25.

Four sequential SOE-PCR amplifications were required to build eachvariable domain gene. The internal-most pair of primers (4 and 5) wereamplified first. The resulting PCR product was gel purified and furtherextended with the next set of external primers (3 and 6). The thirdextension utilized primers 2 and 7, and the final extension utilizedprimers 1 and 8. Each 50 μL reaction contained reaction buffer, 2 unitsof Vent DNA Polymerase (New England Biolabs, Beverly, Mass.),amplification primers at 1 μM each, and dNTPs at 200 μM. Using a GeneAmpPCR 9600 thermocycler (Perkin Elmer, Wellesley, Mass.), samples wereheated for 2 minutes at 94° C., followed by 30 cycles of heating for 30seconds at 94° C., 30 seconds at 55° C., and 30 seconds at 72° C. After30 cycles, the temperature was held constant at 72° C. for 10 minutes toensure complete extension. In each case, the completed PCR reaction mixwas electrophoresed on a 1% agarose gel (Sigma Chemicals, St. Louis,Mo.), and the desired product extracted from a 200 mg gel slice using aQiaquik column (Qiagen, Valencia, Calif.). For the second, third, andfourth reactions, 10 ng of purified product from the preceding reactionwas used as the template. The nucleotide sequences of thehu1G8-A/hu1G8-B V_(L) chain, hu1G8-A V_(H) chain, and hu1G8-B V_(H)chain PCR products are set forth in SEQ. ID. NOs:26, 27, and 28,respectively. Each PCR product contains an engineered Fv regionsurrounded by short stretches of upstream and downstream sequences.

Individually, the purified PCR products were digested with Xba I and XhoI and ligated into one of two expression plasmids (pEE12 for V_(L); pEE6for V_(H)). These plasmids, which contain the Glutamine Synthetase (GS)gene (Lonza Biologics, Slough, UK; Bebbington 1992), had been previouslymodified to contain human κ and IgG1 constant cDNA genes, respectively.At this point, the entire gene (both variable and constant regions) wassequenced. The coding regions of the synthetic genes encoding thehu1G8-B V_(L) and V_(H) chains are set forth in SEQ. ID. NOs:29 and 30,respectively. In the pEE12 light chain plasmid, residue L104, whosecodon is part of the XhoI restriction site, was mutated from leucine tovaline using a Quik-Change kit (Stratagene, San Diego, Calif.) in orderto restore the hu4D5v8 sequence in this region.

A dual chain plasmid was constructed by digesting the pEE6 heavy chainplasmid with BglII and Bam HI to isolate the heavy chain gene andligating this gene into the BamHI site of the pEE12 light chain plasmid.The entire IgG₁ gene was sequenced in both directions to confirm itsidentity. Prior to electroporation the dual chain plasmid was linearizedwith Sal I, filtered through a protein binding membrane to remove therestriction enzyme (Millipore, Bedford, Mass.), ethanol precipitated,and resuspended in sterile water to a concentration of 1 μg/μl.

Example 4 Expression of hu1G8

NS0 murine myeloma cells were electroporated with the dual expressionvector constructed in Example 3, and stable transfectants were selectedby glutamine deprivation as described previously (Yazaki 2001a; Yazaki2004). Secretion of hu1G8 to the cell culture supernatant was measuredby ELISA using goat anti-human IgG (Fc specific) to capture hu1G8constructs and alkaline-phosphatase-conjugated goat anti-human IgG (Fcspecific) antibodies (both from Jackson ImmunoResearch Laboratories) fordetection. The best producing clones were further evaluated by Westernblot for size verification. High producing clones were expanded intoTripleFlasks (Nalge Nunc Int'l, Rochester, N.Y.) or into CELL-PHARM® 100hollow fiber cell culture system (Unisyn Technologies, Hopkinton, Mass.)in 1MDM/1% FBS supplemented with GLUTAMAX® (Invitrogen LifeTechnologies). Expression levels in these clones was at least 10-foldhigher (10-30 μg/ml) than that of ch1G8 in TripleFlasks, validating thepredicted superiority of the hu4D5v8 framework.

Example 5 Purification of ch1G8 and hu1G8

Cell culture supernatants were harvested and passed over a Protein Acolumn (Amersham Biosciences, Piscataway, N.J.) washed with 20 columnvolumes of PBS. Bound antibodies were eluted with 0.1 M glycine (pH 2.7)or with a pH gradient (pH 7.0 to pH 3.0) using 100 mM sodiumphosphate/100 mM sodium citrate buffer. Eluted antibodies wereimmediately neutralized with 1M Tris-HCl (pH 9.0), pooled, and dialyzedagainst 150 nM NaCl/50 mM Tris-HCl (pH 8.0; ch1G8) or PBS (hu1G8) at 4°C. overnight. Antibodies were concentrated using Amicon Centriprep YM-10(Millipore, Billerica, Mass.).

Example 6 Characterization of ch1G8 and hu1G8

Aliquots of purified ch1G8 and hu1G8 were analyzed by SDS-PAGE. ch1G8was found to have a molecular weight of 150 kDa (FIG. 3A, lane 2),similar to that of parental mu1G8 (FIG. 3A, lane 1) under non-reducingconditions. Reducing conditions resulted in separation of the heavy (50kDa) and light (25 kDa) chains. Both hu1G8-A (FIG. 3D, lane 2) andhu1G8-B (FIG. 3D, lane 3) migrated at the same weight as intact mu1G8(FIG. 3D, lane 1) under non-reducing conditions. As with ch1G8, reducingconditions resulted in separation of the heavy (50 kDa) and light (25kDa) chains.

Specific binding of the antibodies was analyzed by flow cytometry onLNCaP-PSCA cells and by Western blot using cell lysates from 293T cellstransiently transfected with pcDNA3.0-PSCA. ch1G8 bound LNCaP-PSCA cellsjust as well as mu1G8 (FIG. 3B), and binding specificity to the antigenwas confirmed by Western blot (FIG. 3C). ch1G8 was found to bind bothbacterial expressed GST-PSCA (lane 1) and 293T-PSCA lysate (lane 3),whereas no binding was observed to 293T control cell lysate (lane 2).The lower molecular weight bands in lanes 1 and 3 represent truncatedprotein and unglycosylated PSCA, respectively. Both humanized antibodiesdemonstrated binding to LNCaP-PSCA that was equivalent to that of mu1G8(FIG. 3E).

The relative binding affinities of the antibodies were measured bycompetitive ELISA using biotinylated mu1G8 as tracer. Wells were coatedwith soluble PSCA fused human IgG1 or mouse IgG2a Fc that had beenexpressed in pEE12 as described above and purified by Protein A affinitychromatography (Poros2.0; PerkinElmer, Wellesley, Mass.). A fixedconcentration of biotinylated parental antibody and increasingconcentrations of non-biotinylated competitors were used as previouslydescribed (Olafsen 2004). Detection of biotinylated antibody was madewith alkaline phosphatase-conjugated streptavidin (JacksonImmunoResearch Laboratories). A reduction in affinity was observed forboth hu1G8 antibodies (FIG. 3F). The K_(D) of mu1G8 was estimated to be5 nM, versus 25 nM for hu1G8-B and 150 nM for hu1G8-A. Due to itssuperior binding affinity, hu1G8-B was selected for in vivo studies.

Example 7 Biodistribution Studies Using mu1G8

Purified mu1G8 was conjugated topisothiocyanatobenzyldiethylenetriamine-pentaacetic acid (MX-DTPA or1B4M-DTPA; Brechbiel 1991) as described previously (Olafsen 2005).Following conjugation, the protein was dialyzed extensively in 0.25 MNH₄OAc (pH 7.0) and concentrated. The MX-DTPA mu1G8 (0.32 mg) wasincubated with 0.60 mCi of carrier free [¹¹¹ In]chloride (MallinckrodtInc., Hazelwood, Mo.) in 0.25 M NH₄OAc (pH 7.0) for 45 minutes at roomtemperature. The reaction was terminated, labeled protein was purifiedby size-exclusion, and labeling efficiency (98.2%) and immunoreactivity(6%), measured by cell-binding to PC3-PSCA, were determined as describedpreviously (Olafsen 2004; Yazaki 2001b). The ¹¹¹In-MX-DTPA mu1G8 wasused to perform a biodistribution study in nude male mice with PSCAxenografts. PC3-PSCA tumor xenografts were established by s.c. injectionof 0.5-1×10⁶ cells resuspended in 50% RPMI/50% MATRIGEL® (BectonDickinson Labware, Bedford, Mass.) in the flanks of the mice. At about14 days post-inoculation, mice bearing xenografts were injected with 3μCi (1.6 μg protein) ¹¹¹In-MX-DTPA mu1G8 via the tail vein. Time pointsanalyzed were 0, 4, 12, 24, 48, 72, and 96 hours. Groups of five micewere euthanized at the different time points and radiouptakes in organswere measured. The percent of the injected dose per gram (% ID/g) withstandard deviations (s.d.) was determined as previously described(Yazaki 2001b; Olafsen 2004). Results are summarized in Table 1, withtumor and normal organ uptake expressed as percent injected dose pergram (% ID/g). Tumor uptake reached a maximum of 17.1 (±6.7) % ID/g at96 hours, with a tumor to blood ratio of 2.1. Hepatic uptake was 8.4(±3.9) % ID/g at 96 hours, whereas the activity in other normal organs(spleen, kidney and lung) was lower.

TABLE 1 Biodistribution of ¹¹¹In-MX-DTPA mu1G8 in nude mice bearingPC3-PSCA xenografts 0 h 4 h 12 h 24 h 48 h 72 h 96 h Organ uptake (%ID/g) Tumor (T) 0.52 2.28 6.87 11.80 13.15 13.75 17.11 (0.17) (0.70)(1.90) (3.07) (6.41) (4.00) (6.67) Blood 31.91 21.86 15.89 13.69 10.847.49 8.01 (2.54) (1.28) (2.15) (0.97) (2.32) (3.30) (3.06) Liver 7.488.41 6.56 7.79 7.39 9.21 8.36 (1.12) (1.78) (1.45) (1.16) (2.32) (3.17)(3.92) Spleen 5.18 3.90 3.53 3.94 4.03 3.02 3.27 (0.77) (0.53) (0.75)(1.13) (0.70) (1.10) (0.91) Kidney 4.52 4.95 4.50 3.95 3.16 2.36 2.25(0.61) (0.74) (1.05) (0.94) (0.68) (0.63) (0.53) Lung 8.60 7.54 6.015.21 4.92 3.23 4.96 (0.61) (1.19) (1.41) (0.88) (0.85) (1.44) (1.58)Carcass 1.89 2.84 3.15 3.35 2.76 2.50 2.40 (0.32) (0.20) (0.44) (0.32)(0.27) (0.36) (0.53) Ratios T:Blood 0.02 0.10 0.43 1.86 1.21 1.84 2.14T:Liver 0.07 0.27 1.05 1.51 1.78 1.49 2.05 T:Kidney 0.12 0.46 1.53 2.994.16 5.83 7.60 Tumor weight 0.089 0.097 0.094 0.092 0.121 0.148 0.114(g) (0.033) (0.030) (0.022) (0.042) (0.036) (0.052) (0.042)

Example 8 Tumor Targeting Using mu1G8 and hu1G8-B

Purified mu1G8 and hu1G8-B (0.2 mg each) were radioiodinated twice withthe positron emitting isotope ¹²⁴I (sodium iodide in 0.02 M NaOH;radionuclide purity >99%) provided by V. G. Khlopin Radium Institute &RITVERC GmbH (St. Petersburg, Russia) as previously described (Kenanova2005). Instant thin layer chromatography using the Monoclonal AntibodyITLC Strips Kit (Biodex Medical Systems, Shirley, N.Y.) was used todetermine the labeling efficiencies. The immunoreactivities weredetermined by cell binding assays. In the first radioiodinationreaction, the labeling efficiencies were 81% and 94%, withimmunoreactivities being 32% and 2%, for the mu1G8 and hu1G8,respectively. In the second reaction, the labeling efficiencies were 90%and 86% with immunoreactivities being 80% and 57% for the mu1G8 andhu1G8, respectively.

PC3-PSCA (antigen positive) and C6 (antigen negative) xenografts wereestablished in nude mice as described above. Four mice were injected inthe tail vein with 130-146 μCi of ¹²⁴I-mu1G8 (specific activity=3.6μCi/μg) or with 136-147 μCi of ¹²⁴I-hu1G8 (specific activity=3.1μCi/μg). Radiolabeled protein from the second labeling reaction wasinjected into male immunodeficient (SCID) mice (Taconic Farms,Germantown, N.Y.) with LAPC-9 (antigen positive) and PC-3 (antigennegative) xenografts. Three mice were injected in the tail vein with127-134 μCi of ¹²⁴I-mu1G8 (specific activity=2.0 μCi/μg) or with 107-111μCi of ¹²⁴I-hu1G8-B (specific activity=1.8 μCi/μg). At the indicatedtime points, mice were anesthetized with 2% isoflurane and imaged usinga Focus microPET scanner (Concorde Microsystems Inc., Knoxville, Tenn.)as previously described (Kenanova 2005). Acquisition time was 10 minutes(1 bed position). Images were reconstructed using a filteredbackprojection (FBP) reconstruction algorithm (Kinahan 1989; Defrise1997) and displayed by AMIDE software (Loening 2003). At the last scantime point (168 hours), one representative SCID mouse from each groupwas also imaged by microCAT™ II tomograph (ImTek Inc., Knoxville, Tenn.)for 10 minutes as described (Chow 2005). The microPET and microCT imageswere then coregistered to yield a single image. After scanning, tumors,liver and kidneys were excised, weighed and counted in a well counter(Willac Wizard 3″, PerkinElmer Life and Analytical Sciences), and afterdecay correction the % ID/g was calculated. In order to determinepositive to negative tumor ratios, regions of interest (ROIs) were drawnas described previously (Sundaresan 2003).

Both antibodies demonstrated specific targeting to the positive tumorand uptake similar to that observed in the biodistribution with¹¹¹In-MX-DTPA mu1G8. At the time of sacrifice (at 94 hours afteradministration), 13.6 (±4.0) % ID/g of the mu1G8 and 12.7 (±1.6) % ID/gof the hu1G8-B was found in the antigen positive tumor (PC3-PSCA),whereas 9.7 (±2.4) % ID/g of mu1G8 and 7.6 (±2.8) % ID/g of hu1G8-B wasfound in the antigen negative tumor (C6). Mice bearing LAPC-9 (antigenpositive) and PC3 (antigen negative) xenografts were imaged every dayfor five days, starting at 72 hours and ending at 168 hours afteradministration (FIG. 6). At 72 hours, most of the activity was seen inthe blood pool and normal tissue. This activity disappears by 168 hours.Activity was also seen in the positive tumor at 72 hours, and at 168hours the highest activity is in the positive tumor as verified by thefused microPET/CT image (far right). The mean positive tumor tobackground ratios were estimated from the images to be 2.2 at 72 hoursand 3.0 at 168 hours for the mu1G8 (n=2), and 1.9 at 72 h and 2.7 at 168hours for the hu1G8-B (n=3). At the time of sacrifice, 5.8 (±0.8) % ID/gof the mu1G8 and 6.6 (±0.9) % ID/g of the hu1G8-B was found in theantigen positive tumor, whereas 2.6 (±0.5) % ID/g of mu1G8 and 3.3(±1.1) % ID/g of hu1G8-B was found in the antigen negative tumor (FIGS.6A and B).

Example 9 Inhibition of Tumor Growth in a Bladder Cancer Model byhu1G8-B

Mice implanted with bladder carcinoma cells were treated with thehu1G8-B. Mice treated with hu1G8-B exhibited markedly lower tumor takeand tumor growth than untreated mice or those treated with 1G8. Thisshows that hu1G8-B is capable of targeting PSCA expressing tumors invivo, and also possesses significant anti-tumor activity in vivo despitedecreased binding affinity compared to 1G8.

SCID mice implanted subcutaneously with 10⁶ SW780 bladder carcinomacells were treated three times per week with either 1G8 or hu1G8-Bantibody at a dosage of 200 μg/mouse via intraperitoneal injection.Control mice received no treatment. A substantial reduction of tumortake was observed in hu1G8-B-treated mice compared to 1G8-treated oruntreated mice (FIG. 7). hu1G8-B treatment resulted in approximately atwo-fold reduction in tumor take/growth. Thus, despite some loss ofbinding affinity compared to parental 1G8 antibody, hu1G8-B possessessignificant anti-tumor activity in vivo that is actually greater thanthat of 1G8.

Example 10 Incorporation of Additional Back Mutations into Humanized 1G8

As stated above in Example 4, hu1G8-B was far more active than hu1G8-Awith regards to antigen binding activity. This suggests that theC-terminal region of the CDR-H2 loop plays a key role in antigenbinding, a conclusion consistent with earlier attempts to humanize anantibody against human protein C(O'Connor 1998). Thus, frameworkresidues that pack against the CDR-H2 loop were scrutinized as potentialtargets for back mutation.

Prior to proceeding, the molecular model of 1G8 was refined. Although1QOK remained the most suitable template for modeling 1G8, several newcrystal structures with individual CDR loops highly similar or identicalto those of 1G8 had been released since the initial model was developed.Comparison of the original model with the new structures suggested thatfor some residues, non-optimal side chain rotamers had been selectedduring the modeling procedure. Furthermore, these new crystal structuresrevealed how several framework residues present in 1G8 influence theconformation of adjacent CDR loops. Based on this, the 1G8 model wasrebuilt and 1G8 was re-humanized using the refined model.

Several methods for superimposing the hu4D5v8 crystal structure with thesecond 1G8 model were evaluated (e.g., superimposing only core packingresidues; superimposing only the V_(H) or V_(L) frameworks, etc.).Differences between the two Fv units present in the unit cell of thehu4D5v8 crystal structure were compared to see if conformationaldifferences that occur as a result of crystal packing interactionsinfluence in any way the outcome of the 1G8 humanization modelingexercise.

Six additional hu1G8-B residues were selected for back mutation to thecorresponding murine residue in an effort to fully restore antigenbinding affinity. Six oligonucleotide primers, ranging in length from 34to 43 oligonucleotides, were used to introduce these back mutations. Thesequences of these six primers are set forth in SEQ. ID. NOs:31-36.hu1G8-B residues L70 (aspartic acid, residue 69 in SEQ. ID. NO:7) andL71 (phenylalanine, residue 70 in SEQ. ID. NO:7) pack against the CDR-L1loop, and in crystal structures with nearly identical CDR-L1 loops thesetwo framework residues influence the conformation of this loop. Based onthis, hu1G8-B residues L70 and L71 were replaced with residues L70 andL71 from 1G8 (serine and tyrosine, residues 69 and 70 in SEQ. ID. NO:1).hu1G8-B residues H48 (valine, residue 48 in SEQ. ID. NO:9) and H49(alanine, residue 49 in SEQ. ID. NO:9), which precede the CDR-H2 loop(H50-H65, residues 50-66 in SEQ. ID. NO:9), were likewise replaced withH48 and H49 from 1G8 (isoleucine and glycine, residues 48 and 49 in SEQ.ID. NO:2). The glycine at residue H49 is particularly important sincethis residue packs against a hu4D5v8 framework residue (H69) thatfollows the CDR-H2 loop. Disruption of this pairing causes major shiftsof the CDR-H2 loop, either toward or away from the CDR-L3 loop (as seenin the original model of hu1G8-B in which hu4D5v8 residues alanine andisoleucine were retained in these positions). For this reason, residueH69 (isoleucine, residue 70 in SEQ. ID. NO:9) was replaced with residueH69 from 1G8 (methionine, residue 70 in SEQ. ID. NO:2). In addition,hu1G8-B residue H66 (arginine, residue 67 in SEQ. ID. NO:9) was replacedwith residue H66 from 1G8 (lysine, residue 67 in SEQ. ID. NO:2) in orderto make the post-CDR-H2 framework region essentially identical in 1G8and the humanized version (residues H67 and H68 are already identical inthe murine and humanized sequences). The amino acid sequences of theresultant hu1G8-C light and heavy chains are set forth in SEQ. ID.NOs:37-38, respectively.

As stated above, the foregoing is merely intended to illustrate variousembodiments of the present invention. The specific modificationsdiscussed above are not to be construed as limitations on the scope ofthe invention. It will be apparent to one skilled in the art thatvarious equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein. All referencescited herein are incorporated by reference as if fully set forth herein.

REFERENCES

-   1. Adams, G. P., Weiner, L. M. 2005. Monoclonal antibody therapy of    cancer. Nat Biotechnol 23:1147-1157.-   2. Bebbington, C., et al. 1992. High-level expression of a    recombinant antibody from myeloma cells using a glutamine synthetase    gene as an amplifiable selection marker. Biotechnology 10:169-175.-   3. Berman, H. M., et al. 2002. The Protein Data Bank. Acta    Crystallogr D Biol Crystallogr 58:899-899-907.-   4. Boehm, M. K., et al. 2000. Crystal structure of the    anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and    a model for antigen binding based on intermolecular contacts.    Biochem J 346 Pt 2:519-528.-   5. Brechbiel, M. W., Gansow, O. A. 1991. Backbone-substituted DTPA    ligands for 90Y radioimmunotherapy. Bioconjug Chem 2:187-194.-   6. Bruggemann, M., et al. 1991. Human antibody production in    transgenic mice: expression from 100 kb of the human IgH locus. Eur    J Immunol 5:1323-1326.-   7. Chow, P. L., Rannou, F. R., Chatziioannou, A. F. 2005.    Attenuation correction for small animal PET tomographs. Phys Med    Biol 50:1837-1850.-   8. Cobleigh, M. A., et al. 1999. Multinational study of the efficacy    and safety of humanized anti-HER2 monoclonal antibody in women who    have HER2-overexpressing metastatic breast cancer that has    progressed after chemotherapy for metastatic disease. J Clin Oncol    17:2639-2648.-   9. Coloma, M. J., et al. 1992. Novel vectors for the expression of    antibody molecules using variable regions generated by polymerase    chain reaction. J Immunol Methods 152:89-104.-   10. Craft, N., et al. 1999. Evidence for clonal outgrowth of    androgen-independent prostate cancer cells from androgen-dependent    tumors through a two-step process. Cancer Res 59:5030-5036.-   11. Defrise, M., et al. 1997. Exact and approximate rebinning    algorithms for 3-D PET data. IEEE Trans Med Imaging 16:145-158.-   12. Eigenbrot, C., et al. 1993. X-ray structures of the    antigen-binding domains from three variants of humanized    anti-p185HER2 antibody 4D5 and comparison with molecular modeling. J    Mol Biol 229:969-995.-   13. Ewert, S., et al. 2003. Biophysical properties of human antibody    variable domains. J Mol Biol 325:531-553.-   14. Foote, J., Winter, G. 1992. Antibody framework residues    affecting the conformation of the hypervariable loops. J Mol Biol    224:487-499.-   15. Geissler, F., et al. 1992. Intracellular catabolism of    radiolabeled anti-mu antibodies by malignant B-cells. Cancer Res    52:2907-2015.-   16. Gu, Z., et al. 2000. Prostate stem cell antigen (PSCA)    expression increases with high gleason score, advanced stage and    bone metastasis in prostate cancer. Oncogene 19:1288-1296.-   17. Gu, Z., et al. 2005. Anti-prostate stem cell antigen monoclonal    antibody 1G8 induces cell death in vitro and inhibits tumor growth    in vivo via a Fc-independent mechanism. Cancer Res 65:9495-9500.-   18. Horton, R. M., et al. 1989. Engineering hybrid genes without the    use of restriction enzymes: gene splicing by overlap extension. Gene    77:61-68.-   19. Jones, P. T., et al. 1986. Replacing the    complementarity-determining regions in a human antibody with those    from a mouse. Nature 321:522-525.-   20. Kenanova, V., et al. 2005. Tailoring the pharmacokinetics and    positron emission tomography imaging properties of    anti-carcinoembryonic antigen single-chain Fv-Fc antibody fragments.    Cancer Res 65:622-631.-   21. Kinahan, P. E., Rogers, J. G. 1989. Analytic 3D image    reconstruction using all detected events. IEEE Trans NS 36:964-968.-   22. Loening, A. M., Gambhir, S. S. 2003. AMIDE: a free software tool    for multimodality medical image analysis. Mol Imaging 2:131-137.-   23. Low, N. M., Holliger, P. H., Winter, G. 1996. Mimicking somatic    hypermutation: affinity maturation. J Mol Biol 260:359-368.-   24. Maynard J., Georgiou, G. 2000. Antibody engineering. Annu Rev    Biomed Eng 2:339-376.-   25. Mendez, M. J., et al. 1997. Functional transplant of megabase    human immunoglobulin loci recapitulates human antibody response in    mice. Nat Genet 2:146-156.-   26. Nanus, D. M., et al. 2003. Clinical use of monoclonal antibody    HuJ591 therapy: targeting prostate specific membrane antigen. J Urol    170:S84-S88; discussion S8-S9.-   27. O'Brien, S., Jones, T. 2003. Humanization of monoclonal    antibodies by CDR grafting. Methods Mol Biol 207:81-100.-   28. O'Connor, S. J., Meng, Y. G., Rezaie, A. R., Presta, L. G. 1998.    Humanization of an antibody against human protein C and    calcium-dependence involving framework residues. Protein Eng    11:321-328.-   29. Olafsen, T., et al. 2004. Characterization of engineered    anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor    targeting. Protein Eng Des Sel 17:315-323.-   30. Olafsen, T., et al. 2005. Optimizing radiolabeled engineered    anti-p185HER2 antibody fragments for in vivo imaging. Cancer Res    65:5907-5916.-   31. Pearson, W. R., Lipman, D. J. 1988. Improved tools for    biological sequence comparison. Proc Natl Acad Sci USA 85:2444-2448.-   32. Reiter, R. E., et al. 1998. Prostate stem cell antigen: a cell    surface marker overexpressed in prostate cancer. Proc Natl Acad Sci    USA 95:1735-1740.-   33. Reiter, R. E., et al. 2000. Coamplification of prostate stem    cell antigen (PSCA) and MYC in locally advanced prostate cancer.    Genes Chromosomes Cancer 27:95-103.-   34. Saffran, D. C., et al. 2001. Anti-PSCA mAbs inhibit tumor growth    and metastasis formation and prolong the survival of mice bearing    human prostate cancer xenografts. Proc Natl Acad Sci USA    98:2658-2663.-   35. Schroff, R. W., Foon, K. A., Beatty, S. M., Oldham, R. K., and    Morgan, A. C. Jr. 1985. Human anti-murine responses in patients    receiving monoclonal antibody therapy. Cancer Res 45:879-885.-   36. Shawler, D. L., Bartholomew, R. M., Smith, L. M., and    Dillman, R. O. 1985. Human immune responses to multiple injections    of murine monoclonal immunoglobulins. J Immunol 135:1530-1535.-   37. Smith-Jones, P. M. 2004. Radioimmunotherapy of prostate cancer.    Q J Nucl Med Mol Imaging 48:297-304.-   38. Sundaresan, G., et al. 2003. 124I-labeled engineered anti-CEA    minibodies and diabodies allow high-contrast, antigen-specific    small-animal PET imaging of xenografts in athymic mice. J Nucl Med    44:1962-1969.-   39. Winter, G., Griffiths, A. D., Hawkins, R. E.,    Hoogenboom, H. R. 1994. Making antibodies by phage display    technology. Annu Rev Immunol 12:433-455.-   40. Worn, A., Pluckthun, A. 1999. Different equilibrium stability    behavior of ScFv fragments: identification, classification, and    improvement by protein engineering. Biochemistry 38:8739-8750.-   41. Wu, A. M., et al. 2001. Multimerization of a chimeric anti-CD20    single-chain Fv-Fc fusion protein is mediated through variable    domain exchange. Protein Eng 14:1025-1033.-   42. Wu, A. M., Senter, P. D. 2005. Arming antibodies: prospects and    challenges for immunoconjugates. Nat Biotechnol 23:1137-1146.-   43. Xu, F. J., et al. 1997. Radioiodinated antibody targeting of the    HER-2/neu oncoprotein. Nucl Med Biol 24:451-459.-   44. Yazaki, P. J., et al. 2001a. Mammalian expression and hollow    fiber bioreactor production of recombinant anti-CEA diabody and    minibody for clinical applications. J Immunol Methods 253:195-208.-   45. Yazaki, P. J., et al. 2001b. Tumor targeting of radiometal    labeled anti-CEA recombinant T84.66 diabody and t84.66 minibody:    comparison to radioiodinated fragments. Bioconjug Chem 12:220-228.-   46. Yazaki, P. J., et al. 2004. Humanization of the anti-CEA T84.66    antibody based on crystal structure data. Protein Eng Des Sel    17:481-489.

What is claimed is:
 1. A humanized antibody comprising: a) a light chainvariable region comprising the amino acid sequence of SEQ ID NO:7 and b)a heavy chain variable region comprising the amino acid sequence of SEQID NO:9, wherein said humanized antibody binds prostate stem cellantigen (PSCA), and wherein administration of said humanized antibody toa subject diagnosed with prostate cancer decreases tumor growth to agreater extent than treatment with the murine 1G8 monoclonal antibodyproduced by the hybridoma deposited as ATCC No. HB-12612.
 2. Thehumanized antibody of claim 1, wherein said antibody has an affinityconstant for prostate stem cell antigen (PSCA) of at least about2.5×10⁸.
 3. The humanized antibody of claim 1, further comprising aconjugate.
 4. The humanized antibody of claim 3, wherein said conjugateis selected from the group consisting of a toxin, a cytokine, achemotherapeutic agent, and a radiolabel.